An adjuvant is an agent administered to potentiate an immune response to an antigen and/or to modulate an immune response toward a desired immune response. An endogenous adjuvant is a compound or molecule naturally occurring within a cell or tissue that likewise enhances an immune response by stimulating innate immunity, thus possessing the capacity to potentiate an effect of some triggering event or agent. Endogenous adjuvants play a central role in alerting the immune system to potential danger and in promoting response to infection, transplantation, tumor, and autoimmunity.
Vaccines attempt to safely elicit an immunity to pathogens that is ideally robust, protective and long-lived. However, current formulations of many subunit vaccines provide weaker and shorter-lived immunity than natural infection. While it is clear that adjuvants can be used to boost immunity, the adjuvants that are permitted in licensed vaccines are limited. Alum, a mixture of aluminum salts, was the first vaccine adjuvant that was widely utilized in vaccine preparations. It was the only vaccine adjuvant in use in the United States until 2009, when the U.S. Food and Drug Administration approved Cervarix, a human papillomavirus vaccine that contains an adjuvant designated as AS04. The AS04 adjuvant is a mixture of alum and a bacterial lipid (fat) molecule that has been modified so that it does not cause disease.
Alum, however, is a weak adjuvant and one that biases responses to effector responses (Th2) that are not protective against many pathogens. Endogenous adjuvants generally have not been evaluated for their potential use in vaccines. In theory, they may allow vaccinations to safely mimic the pathway that naturally triggers immunity to many pathogens. These agents also promote CD8+ T cell immune responses, which are important in immunity to many pathogens, such as viruses and tumors, but not elicited by most subunit vaccines (Rock et al. in Springer Seminars in Immunopathology (2005) 26:231-246).
Purine Nucleoside Phosphorylase (PNP) is an enzyme involved in purine metabolism. PNP metabolizes inosine and deoxyinosine into hypoxanthine, and guanosine and deoxyguanosine into guanine; in each case creating (deoxy) ribose phosphate. PNP-deficient patients exhibit significantly higher levels of plasma nucleosides including inosine, deoxyinosine, guanosine and deoxyguanosine when compared to PNP-normal subjects (Markert in Immunodeficiency Review (1991) 3:45-81), and further exhibit elevated levels of erythrocyte deoxyguanosine triphosphate (dGTP) and nicotinamide adenine dinucleotide (NAD). PNP-deficient patients inevitably manifest an immunodeficiency problem affecting T-cells and B-cells. Plasma deoxyguanosine (the only clinically measurable nucleoside) and intracellular dGTP are elevated in patients treated with PNP inhibitors (Bantia and Kilpatrick in Current Opinions in Drug Discovery & Development (2004) 7: 243-247). Deoxyguanosine was also elevated in mouse plasma after treatment with PNP inhibitor (Bantia et al. in International Immuno-pharmacology (2001) 1:1199-1210 and (2010) 10:784-790).
A major source of nucleoside pools comes from the breakdown of RNA and DNA during normal cell turnover, cellular injury or cell death due to infection. Normally the nucleosides deoxyguanosine, inosine, deoxyinosine, and guanosine are present at very low to undetectable levels in the plasma because PNP is an extremely efficient catalyst and rapidly breaks down inosine and deoxyinosine to hypoxanthine, and guanosine and deoxyguanosine to guanine and sugar 1-phosphate. In the presence of a PNP inhibitor or due to a PNP deficiency, however, these nucleosides become elevated. Guanosine analogs like isatoribine (7-thia, 8-oxoguanosine), loxorabine (7-allyl, 8-oxo guanosine) and others have been shown to be immuno-potentiators, demonstrating antiviral, antibacterial and anticancer effects in animal models (Smee et al. in Antimicrobial Agents and Chemotherapy (September 1989) 1487-1492; Stewart et al. in J. Interferon Cytokine Research (2012) 32(1):46-51; also in Poult Science (2012) 91(4):1038-1042; Pope et al. in Cell Immunol. (1995) 162(2):333-339).
ANA773, an oral pro-drug of isatoribine, has been demonstrated to induce endogenous interferon-a (IFN-a) of multiple subtypes in healthy volunteers. In clinical trials of chronically HCV infected patients, ANA773 demonstrated a dose-dependent reduction in HCV RNA (Bergmann et al. in Aliment Pharmacol Ther (2011) 34:443-453; International patent number WO2005025583A2).
In-vitro studies with guanosine analogs have shown activation of immune cells such as dendritic cells and natural killer cells to produce ifn-gamma, which is mediated through Toll-Like Receptor 7 (TLR7). Toll-like receptors (TLRs) have been established as a family of pathogen recognition receptors (PRR) that initiate the innate immune response. In addition to TLRs there are other PRR's, for example retinoic acid inducible gene I (RIGI) like receptors (RLR), nucleotide binding oligomerization domain (NOD)-like receptors (NLR) and c-type lectin receptors (CLR). Direct or indirect stimulation of TLRs and other PRRs causes the release of multiple cytokines including type 1 and type 2 interferons, the induction of pathways and enzymes that destroy intracellular pathogens, the activation of a variety of cellular responses, and the priming of the adaptive response by activation of immature dendritic cells, inducing their differentiation into professional antigen-presenting cells. At least eleven different TLR genes have been identified in humans. It appears that through stimulation of innate immunity by activating TLR, it is possible to prevent or reverse otherwise lethal viral infections in various acute infection models in mice.
Methyl inosine monophosphate, a particular inosine analog, has also shown immune enhancing effects and demonstrated antiviral and antibacterial effects (Mishin et al. in Antiviral Research (2006) 71:64-68).
In addition to the accumulation of nucleosides in the presence of PNP inhibitor, deoxyguanosine is converted to dGTP in lymphocytes and erythrocytes. dGTP could potentially stimulate the immune system through activation of PRR's in the presence of an antigen similar to what has been observed with ATP. Although the mechanism is not clear, PNP deficient patients also demonstrate increase in NAD levels. NAD may also serve as danger signal and activate the immune system (Haag et al., Purinergic Signalling (2007) 3: 71-81).
Based on the role of PNP in purine catabolism, the present investigators hypothesize that effective inhibition of PNP may elevate nucleosides, inosine, deoxyinosine, guanosine deoxyguanosine and nucleotides dGTP and NAD levels in a subject, as is seen in PNP-deficient patients and PNP-deficient mice (FIG. 1); however contrary to expectations based on the immuno-compromised clinical phenotype of the PNP-deficient patient, the present investigators have discovered that PNP inhibition in a PNP-normal patient results in an immune-potentiating effect.
There remains a need in the art for methods for preventing and treating diseases which exploit the natural endogenous adjuvant response. Controlling the levels of endogenous adjuvants provides a novel mechanism to exploit for enhancing immunogenicity of an antigen and augmenting potency of vaccines and cancer immunotherapies. Further, identification of endogenous adjuvants, triggered in response to certain pathogens, may provide novel exogenous adjuvants, which may thereafter be administered exogenously to enhance an immune response and augment the potency of vaccines and cancer immunotherapies.